Methods of treatment for cholestatic and fibrotic diseases

ABSTRACT

The present invention relates to the compound [2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate (Nitazoxanide) or 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide (Tizoxanide) for treating cholestatic and fibrotic diseases.

SEQUENCE LISTING

The Instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Dec. 14, 2017, is named“1121_006409-US3_ST25.txt” and is 1 KB in size.

TECHNICAL FIELD

The present invention relates to the field of medicine, in particular tothe treatment of cholestatic or fibrotic diseases.

BACKGROUND

Abnormal and exaggerated deposition of extracellular matrix is thehallmark of all fibrotic diseases, including liver, pulmonary, kidney orcardiac fibrosis. The spectrum of affected organs, the progressivenature of the fibrotic process, the large number of affected persons,and the absence of effective treatment pose an enormous challenge whentreating fibrotic diseases.

In an attempt to propose new therapeutic strategies for the treatment offibrotic diseases, the inventors found that the compound2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate (Nitazoxanide orNTZ), a synthetic antiprotozoal agent, also shows potent antifibroticproperties. Moreover, the evaluation of NTZ in a liver injury modelrevealed its capacity to reduce circulating bile acid concentration,thus reflecting its potential to treat both cholestatic (such as PBC andPSC) and fibrotic diseases.

NTZ, first described in 1975 (Rossignol and Cavier, 1975), was shown tobe highly effective against anaerobic protozoa, helminths, and a widespectrum of microbes including both anaerobic and aerobic bacteria(Rossignol and Maisonneuve, 1984; Dubreuil, Houcke et al., 1996;Megraudd, Occhialini et al., 1998; Fox and Saravolatz, 2005; Pankuch andAppelbaum, 2006; Finegold, Molitoris et al., 2009). It was first studiedin humans for the treatment of intestinal cestodes (Rossignol andMaisonneuve, 1984) and it is now licensed in the United States (Alinia®,Romark laboratories) for the treatment of diarrhea caused by theprotozoan parasites Crystosporidium parvum and Giardia intestinalis. NTZhas also been widely commercialized in Latin America and in India whereit is indicated for treating a broad spectrum of intestinal parasiticinfections (Hemphill, Mueller et al., 2006). The proposed mechanism ofaction by which NTZ exerts its antiparasitic activity is through theinhibition of pyruvate:ferredoxin oxidoreductase (PFOR) enzyme-dependentelectron transfer reactions that are essential for anaerobic metabolism(Hoffman, Sisson et al., 2007). NTZ also exhibits activity againstMycobacterium tuberculosis, which does not possess a homolog of PFOR,thus suggesting an alternative mechanism of action. Indeed, it was shownthat NTZ can also act as an uncoupler disrupting membrane potential andintra-organism pH homeostasis (de Carvalho, Darby et al., 2011).

The pharmacological effects of NTZ are not restricted to itsantiparasitic activities and in recent years, several studies revealedthat NTZ can also confer antiviral activity (Di Santo and Ehrisman,2014; Rossignol, 2014). NTZ Interferes with the viral replication bydiverse ways including a blockade in the maturation of hemagglutinin(influenza) or VP7 (rotavirus) proteins, or the activation of theprotein PKR involved in the innate immune response (for a review, see(Rossignol, 2014)). NTZ was also shown to have broad anticancerproperties by interfering with crucial metabolic and prodeath signalingpathways (Di Santo and Ehrisman, 2014)

In this invention, using a phenotypic screening assay to identifypotential antifibrotic agents, it was discovered that NTZ or its activemetabolite Tizoxanide (or TZ) interferes with the activation of hepaticstellate cells (HSC), which play a key role in the development ofhepatic fibrosis. This effect was totally unexpected in view of theproperties previously reported for these molecules. Moreover, NTZ and TZwere shown to interfere with the activation of stimulated fibroblastsderived from other organs such as heart, lung and intestines. Theantifibrotic properties of NTZ was further confirmed in a preclinicalmodel of liver disease (CDAAc diet-induced NASH) by showing significantreduced levels of hepatic collagen and fibrosis. In addition to itsantifibrotic activity, NTZ was also shown to reduce circulating bileacid concentration in a CCl4-induced liver injury model. NTZ and TZ thusappear as compounds of interest for the treatment of cholestaticdiseases and diverse types of fibrotic diseases.

SUMMARY OF INVENTION

The present invention relates to compound[2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate (Nitazoxanide)or its active metabolites 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide(Tizoxanide), or Tizoxanide glucuronide (TZG), or a pharmaceuticallyacceptable salt thereof, for use in a method for the treatment of acholestatic or fibrotic disease.

In a particular embodiment, the fibrotic disorder is selected in thegroup consisting of liver, gut, kidney, skin, epidermis, endodermis,muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervoussystem, testis, penis, ovary, adrenal gland, artery, vein, colon,intestine (e.g. small intestine), biliary tract, soft tissue (e.g.mediastinum or retroperitoneum), bone marrow, joint, eye and stomachfibrosis. In a further particular embodiment, the fibrotic disorder isselected in the group consisting of liver, kidney, skin, epidermis,endodermis, muscle, tendon, cartilage, heart, pancreas, lung, uterus,nervous system, testis, ovary, adrenal gland, artery, vein, colon,intestine (e.g. small intestine), biliary tract, soft tissue (e.g.mediastinum or retroperitoneum), bone marrow, joint and stomachfibrosis. In a further particular embodiment, the fibrotic disorder isselected in the group consisting of liver, gut, lung, heart, kidney,muscle, skin, soft tissue, bone marrow, intestinal, and joint fibrosis.In yet another embodiment the fibrotic disorder is selected in the groupconsisting of non-alcoholic steatohepatitis (NASH), pulmonary fibrosis,idiopathic pulmonary fibrosis, skin fibrosis, eye fibrosis (such ascapsular fibrosis), endomyocardial fibrosis, mediastinal fibrosis,myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (acomplication of coal workers' pneumoconiosis), proliferative fibrosis,neoplastic fibrosis, lung fibrosis consecutive to chronic inflammatoryairway disease (COPD, asthma, emphysema, smoker's lung, tuberculosis),alcohol or drug-induced liver fibrosis, liver cirrhosis,infection-induced liver fibrosis, radiation or chemotherapeutic-inducedfibrosis, nephrogenic systemic fibrosis, Crohn's disease, ulcerativecolitis, keloid, old myocardial Infarction, scleroderma/systemicsclerosis, arthrofibrosis, some forms of adhesive capsulitis, chronicfibrosing cholangiopathies such as Primary Sclerosing Cholangitis (PSC),Primary Biliary Cholangitis (PBC), biliary atresia, familialintrahepatic cholestasis type 3 (PFIC3), peri-implantational fibrosisand asbestosis.

According to a particular embodiment of the invention, thecholestestatic disease is selected in the group consisting of primarybiliary cholangitis (PBC), primary sclerosing cholangitis (PSC),Intrahepatic Cholestasis of Pregnancy, Progressive Familial IntrahepaticCholestasis, Biliary atresia, Cholelithiasis, Infectious Cholangitis,Cholangitis associated with Langerhans cell histiocytosis. Alagillesyndrome, Nonsyndromic ductal paucity, Drug-induced cholestasis, andTotal parenteral nutrition-associated cholestasis. In a particularembodiment, the cholestatic disease is PBC.

According to another aspect, the invention relates to a pharmaceuticalcomposition comprising a compound selected from NTZ or TZ(G), or apharmaceutically acceptable salt of NTZ or TZ(G), for use in a methodfor treating a cholestatic or fibrotic disorder, wherein saidcompound(s) is (are) the only active ingredient(s) in the composition.

According to another aspect, the invention relates to the compound orthe pharmaceutical composition as defined above, for use in thetreatment of a fibrotic disorder in combination with at least onetherapeutically active agent with known antifibrotic activity selectedfrom pirfenidone or receptor tyrosine kinase inhibitors (RTKIs) such asNintedanib, Sorafenib and other RTKIs, or angiotensin II (AT1) receptorblockers, or CTGF inhibitor, or any antifibrotic compound susceptible tointerfere with the TGFβ- and BMP-activated pathways including activatorsof the latent TGFβ complex such as MMP2, MMP9, THBS1 or cell-surfaceintegrins, TGFβ receptors type I (TGFBRI) or type II (TGFBRII) and theirligands such as TGFβ, Activin, inhibin, Nodal, anti-Müllerian hormone,GDFs or BMPs, auxiliary co-receptors (also known as type III receptors),or components of the SMAD-dependent canonical pathway includingregulatory or inhibitory SMAD proteins, or members of theSMAD-independent or non-canonical pathways including various branches ofMAPK signaling, TAK1, Rho-like GTPase signaling pathways,phosphatidylinositol-3 kinase/AKT pathways. TGFβ-induced EMT process, orcanonical and non-canonical Hedgehog signaling pathways including Hhligands or target genes, or any members of the WNT, or Notch pathwayswhich are susceptible to influence TGFβ signaling.

The invention further relates to the compound or pharmaceuticalcomposition as defined above, for use in combination with at least onetherapeutically active agent selected from JAK/STAT inhibitors, otheranti-inflammatory agents and/or immunosuppressant agents.

According to a particular embodiment, the therapeutically active agentis selected from glucocorticoids, NSAIDS, cyclophosphamide,nitrosoureas, folic acid analogs, purine analogs, pyrimidine analogs,methotrexate, azathioprine, mercaptopurine, ciclosporin, myriocin,tacrolimus, sirolimus, mycophenolic acid derivatives, fingolimod andother sphingosine-1-phosphate receptor modulators, monoclonal and/orpolyclonal antibodies against such targets as proinflammatory cytokinesand proinflammatory cytokine receptors, T-cell receptor, integrins.

The invention further relates to a pharmaceutical composition comprising

-   -   NTZ or a pharmaceutically acceptable salt of NTZ; and    -   TZ(G), or a pharmaceutically acceptable salt of TZ(G).

The invention also relates to a kit-of-parts comprising:

-   -   NTZ or a pharmaceutically acceptable salt of NTZ; and    -   TZ(G) or a pharmaceutically acceptable salt of TZ(G).

According to a particular embodiment, in each aspects and embodimentsdescribed herein, NTZ or TZ, or a pharmaceutically acceptable salt ofNTZ or TZ is used.

DESCRIPTION OF THE FIGURES AND TABLES

Abbreviations Used in the Figures, in the Tables, and in the Text:

α-SMA: alpha Smooth Muscle Actin

BMP: Bone Morphogenetic Protein

cDNA: Complementary Deoxyribonucleotide Acid

COL1A1: Collagen, type 1, Alpha 1

CDAA: Choline-Deficient L-Amino Acid-defined diet

CDAAc: Choline-Deficient L-Amino Acid-defined diet supplemented withcholesterol

CHOL: cholesterol

CSAA: Choline Supplemented L-Amino Acid-defined diet

CYPA: Cyclophilin A

DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine

DMSO: Dimethyl sulfoxide

ELISA: Enzyme-Linked Immunosorbent Assay

EMT: Epithelial-mesenchymal transition

DTT: Dithiothreitol

FBS: Fetal Bovine Serum

FDA: Food and Drug Administration

GDF: Growth Differentiation Factors

Hh: Hedgehog

hHSC: Human Hepatic Stellate Cells

HSC: Hepatic Stellate Cells

IC₅₀: Half maximal Inhibitory Concentration

InMyoFib: Intestinal Myofibroblasts

MMP2: Matrix Metallopeptidase 2

MMP9: Matrix Metallopeptidase 9

μl: microliter

NHLF: Normal Human Lung Fibroblasts

NTZ: Nitazoxanide

PBC: Primary Biliary Cholangitis

PBS: Phosphate Buffer Saline

PSC: Primary Sclerosing Cholangitis

qPCR: Quantitative Polymerase Chain Reaction

pMol: picomoles

rhFGF: recombinant human basic Fibroblast Growth Factor

RNA: Ribonucleic Acid

RT: Reverse Transcriptase

SmBM: Smooth Muscle cell Basal Medium

SteCGS: Stellate Cell Growth Supplement

STeCM: Stellate Cell Medium

TBA: Total Bile Acids

TGFβ1: Tumor Growth Factor beta 1

TGFBRI: TGFb type I receptor

TGFBRII: TGFb type II receptor

THBS1: Thrombospondine 1

TMB: Tetramethylbenzidine

TZ: Tizoxanide

TZG: Tizoxanide glucuronide

TZ(G): TZ or TZG

FIG. 1. Nitazoxanide and its Metabolite Tizoxanide Inhibit TGFβ1-InducedExpression of α-SMA Protein in Human HSC

Serum-deprived HSC were preincubated for 1 hour with NTZ (A) or TZ (B)before the activation with the profibrogenic cytokine TGFβ1 (1 ng/ml).After 48 hours of incubation, the expression of α-SMA was measured byELISA. The obtained values were transformed into percentage inhibitionover TGFβ1 control. Data are presented as mean (triplicates)±standarddeviation (SD). Statistical analyses were performed by one-way ANOVAfollowed by Bonferroni post-hoc tests, using Sigma Plot 11.0 software.[*: p<0.05; **: p<0.01; ***: p<0.001 (comparison versus TGFβ1 1 ng/mLgroup)]. The curve fitting and the calculation of half maximalinhibitory concentration (IC₅₀) were performed with XLFit software5.3.1.3.

FIG. 2. Nitazoxanide and its Metabolite Tizoxanide Reduce COL1A1Transcripts in TGFβ1-Induced Human HSC

Serum-deprived HSC were preincubated for 1 hour with NTZ (A) or TZ (B)before the activation with TGFβ1 (1 ng/ml). After 24 hours ofincubation, the expression of COL1A1 was measured by RT-qPCR. Expressionvalues were transformed into fold induction over TGFβ1 control. Data arepresented as mean (triplicates)±standard deviation (SD). Statisticalanalyses were performed by one-way ANOVA followed by Bonferroni post-hoctests, using Sigma Plot 11.0 software. [*: p<0.05; **: p<0.01; ***:p<0.001 (comparison versus TGFβ1 1 ng/mL group)].

FIG. 3: NTZ (A) or TZ (B) Inhibit TFGβ1-Induced Expression of α-SMAProtein in Rat HSCs.

NTZ (A) or TZ (B) were added to serum-deprived rat HSC (rHSC) 1 hourbefore the activation with TGFβ1 (3 ng/ml). After 48 hours ofincubation, the expression of α-SMA was measured by ELISA. The obtainedvalues were transformed into percentage inhibition over TGFβ1 control.Data are presented as mean (triplicates)±standard deviation (SD).Statistical analyses were performed by one-way ANOVA followed byBonferroni post-hoc tests, using Sigma Plot 11.0 software. [*: p<0.05;**: p<0.01; ***: p<0.001 (comparison versus TGFβ1 3 ng/mL group)].

FIG. 4: NTZ (A) or TZ (B) Inhibit TFGβ1-Induced Expression of α-SMAProtein in Human Lung Fibroblasts.

NTZ (A) or TZ (B) was added to serum-deprived lung fibroblasts (NHLF) 1hour before the activation with TGFβ1 (1 ng/ml). After 48 hours ofincubation, the expression of α-SMA was measured by ELISA. The obtainedvalues were transformed into percentage inhibition over TGFβ1 control.Data are presented as mean (triplicates)±standard deviation (SD).Statistical analyses were performed by one-way ANOVA followed byBonferroni post-hoc tests, using Sigma Plot 11.0 software. [*: p<0.05;**: p<0.01; ***: p<0.001 (comparison versus TGFβ1 1 ng/mL group)].

FIG. 5: NTZ (A) or TZ (B) Inhibit TFGβ1-Induced Expression of α-SMAProtein in Human Cardiac Fibroblasts.

NTZ (A) or TZ (B) were added to serum-deprived cardiac fibroblasts(NHCF) 1 hour before the activation with TGFβ1 (3 ng/ml). After 48 hoursof incubation, the expression of α-SMA was measured by ELISA. Theobtained values were transformed into percentage inhibition over TGFβ1control. Data are presented as mean (triplicates)±standard deviation(SD). Statistical analyses were performed by one-way ANOVA followed byBonferroni post-hoc tests, using Sigma Plot 11.0 software. [*: p<0.05;*: p<0.01; ***: p<0.001 (comparison versus TGFβ1 3 ng/mL group)].

FIG. 6: NTZ (A) or TZ (B) Inhibit TFGβ1-Induced Expression of α-SMAProtein in Human Intestinal Fibroblasts.

NTZ (A) or TZ (B) were added to serum-deprived intestinal fibroblasts(InMyoFib) 1 hour before the activation with TGFβ1 (3 ng/ml). After 48hours of incubation, the expression of α-SMA was measured by ELISA. Theobtained values were transformed into percentage inhibition over TGFβ1control. Data are presented as mean (triplicates)±standard deviation(SD). Statistical analyses were performed by one-way ANOVA followed byBonferroni post-hoc tests, using Sigma Plot 11.0 software. [*: p<0.05;**: p<0.01; ***: p<0.001 (comparison versus TGFβ1 3 ng/mL group)].

FIG. 7: The Chronic Oral Administration of Nitazoxanide (10 mg/kd/day)Prevents the CDAA-Induced Collagen Storage in the Liver of C57Bl/6JMice.

6 week-old C57Bl/6 mice were fed a control (CSAA) diet, CDAA+1% CHOL(CDAAc) diet, or CDAAc diet supplemented with NTZ 10 mg/kg/day for 12weeks. After the sacrifice, the hepatic collagen content was determined.Data are presented as mean±standard deviation (SD). Statistical analyseswere performed by a student t-test using Sigma Plot 11.0 software: CSAAvs CDAAc (#: p<0.05; ##: p<0.01; ###: p<0.001) and CDAAc vs NTZ 10mg/kg/day (*: p<0.05; **: p<0.01; ***: p<0.001).

FIG. 8: The Chronic Oral Administration of Nitazoxanide (10 mg/kg/day)Prevents the CDAAc Diet-Induced Fibrosis in the Liver of C57Bl/6J Mice.

6 week-old C57Bl/6 mice were fed a control (CSAA) diet, CDAAc diet, orCDAAc diet supplemented with NTZ 10 mg/kg/day for 12 weeks. After thesacrifice, the hepatic fibrosis area was determined. Data are presentedas mean±standard deviation (SD). Statistical analyses were performed bya student t-test using Sigma Plot 11.0 software: CSAA vs CDAAc (#:p<0.05; ##: p<0.01; ###: p<0.001) and CDAAc vs NTZ 10 mg/kg/day (*:p<0.05: **: p<0.01: ***: p<0.001).

FIG. 9: The Chronic Oral Administration of Nitazoxanide PreventsCCl4-Induced Levels of Circulating TBA Concentration.

250-275 g rats were intraperitoneally injected with olive oil (ctrlgroup) or with CCl4 emulsified in olive oil (CCl4:olive oil 1:2 v/v,final CCl4 concentration: 2 ml/kg) twice weekly for 3 weeks.Concomitantly, the olive oil injected group was placed on control dietwhile the CCl4 injected groups were placed on control diet or dietsupplemented with NTZ 10 mg/kg/day or 30 mg/kg/day. After the sacrifice,circulating TBA concentration was determined. Data are presented asmean±standard deviation (SD). Statistical analyses were performed by astudent t-test, using Sigma Plot 11.0 software: Olive Oil vs CCl4 (#:p<0.05; ##: p<0.01; ###: p<0.001) and CCl4 vs NTZ (*: p<0.05; **:p<0.01; ***: p<0.001).

DETAILED DESCRIPTION OF THE INVENTION

In the experimental part of the present application, it is shown thatcompounds [2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate(Nitazoxanide) and 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide(Tizoxanide) have anti-fibrotic properties in several models offibrosis. Moreover, it is shown that NTZ, or its active metabolite TZ,have the capacity to prevent the occurrence of altered levels ofcirculating bile acids in a model of liver injury, showing the abilityof NTZ and TZ to treat cholestatic diseases. Accordingly, the presentinvention relates to novel therapeutic uses of compound NTZ or of anactive metabolite thereof such as TZ or TZG.

In particular, the present invention relates to the compound NTZ orTZ(G), or a pharmaceutically acceptable salt of NTZ or TZ(G), for use ina method for the treatment of a cholestatic or fibrotic disorder. Theinvention relates also to a pharmaceutical composition comprising NTZ orTZ(G), or a pharmaceutically acceptable salt thereof, for use in amethod for the treatment of a cholestatic or fibrotic disorder.Furthermore, the invention relates to the use of NTZ or TZ(G), or apharmaceutically acceptable salt thereof, for the manufacture of amedicament useful for the treatment of a cholestatic or fibroticdisorder. The invention also relates to a pharmaceutical compositioncomprising NTZ or TZ(G), or a pharmaceutically acceptable salt thereof.The pharmaceutical composition according to the invention is useful fortreating a cholestatic or fibrotic disorder.

Although the causative agents or initiating events of fibrotic disordersare quite diverse and their pathogenesis is variable, a common featurein affected tissues is the presence of large numbers of activatedfibroblasts called myofibroblasts ((Rosenbloom, Mendoza et al., 2013)).Fibrotic stimulus such as TGFβ1 can induce differentiation offibroblasts to myofibroblasts (Leask and Abraham, 2004; Leask, 2007).Myofibroblasts are metabolically and morphologically distinctivefibroblasts whose activation and proliferation play a key role indevelopment of the fibrotic response. Furthermore, these myofibroblastsdisplay unique biological functions including expression of proteinsinvolved in extracellular matrix formation such as different forms ofcollagen, fibronectin and other ECM proteins. The induction of α-smoothmuscle actin (α-SMA) expression is a recognized hallmark of quiescentfibroblast to activated myofibroblast differentiation and can be used asa physiological read-out to evaluate the potency of the drugs thatinterfere with the fibrotic process. Tumor Growth β factors, andespecially the Tumor Growth Factor beta 1 (TGFβ1) are recognizedphysiological signals that induce the phenotypic transformation offibroblasts into profibrotic myofibroblasts that express high levels ofα-SMA and high levels of extracellular matrix proteins, which are thensecreted and form the fibrotic scar tissue.

Moreover, it is known that the proliferation and the activation offibroblasts is responsible for the production of several connectivetissue components (for example, collagens, elastin, proteoglycans, andhyaluronan) that constitute the extracellular matrix (Kendall andFeghali-Bostwick, 2014).

Unexpectedly, NTZ but also its active metabolite TZ, reveal antifibroticproperties since these compounds dose-dependently reduced the level ofα-SMA in TGFβ-induced hepatic stellate cells and in primary fibroblastsfrom other organs. Furthermore, treatment with NTZ or TZ also repressedcollagen (Col1a1) expression in TGFβ activated rat HSC, which confirmsantifibrotic properties of both molecules. The antifibrotic activity ofNTZ, or its metabolite TZ was also demonstrated in vivo using a model ofCDAAc-induced liver fibrosis, in which reduced hepatic collagen contentand diminished fibrosis area were exemplified. Moreover, in theCCl4-Induced liver injury model, it was shown that NTZ, or its activemetabolite TZ, could prevent the induction of circulating bile acidlevels, which represent a marker of cholestatic diseases.

NTZ, TZ and TZG to be used according to the invention have the followingFormula (I), (II) and (III) respectively:

NTZ and TZ were known for their antiparasitic and antiviral activities,but the prior art does not teach that NTZ, TZ and TZG haveanticholestatic and anti-fibrotic effects.

The inventors have demonstrated in a new and inventive way that thesecompounds have a therapeutic effect in the treatment of cholestasis orfibrosis.

Accordingly, the invention relates to compound NTZ or TZ(G), or apharmaceutically acceptable salt of NTZ or TZ(G), for use in a methodfor the treatment of a cholestatic or fibrotic disorder.

In a further aspect, the invention relates to NTZ or TZ(G), or apharmaceutically acceptable salt of NTZ or TZ(G), for use in theinhibition of proliferation and/or activation of fibroblasts. As isknown in the art, fibroblasts are responsible for the production ofcollagen fibers or other connective tissue components of theextracellular matrix.

According to the present invention, the terms “fibrosis”, “fibroticdisease”, “fibrotic disorder” and declinations thereof denote apathological condition of excessive deposition of fibrous connectivetissue in an organ or tissue. More specifically, fibrosis is apathological process, which includes a persistent fibrotic scarformation and overproduction of extracellular matrix by the connectivetissue, as a response to tissue damage. Physiologically, the deposit ofconnective tissue can obliterate the architecture and function of theunderlying organ or tissue.

According to the present invention, the fibrosis or fibrotic disordermay be associated with any organ or tissue fibrosis. Illustrative,non-limiting examples of particular organ fibrosis include liver, gut,kidney, skin, epidermis, endodermis, muscle, tendon, cartilage, heart,pancreas, lung, uterus, nervous system, testis, penis, ovary, adrenalgland, artery, vein, colon, intestine (e.g. small intestine), biliarytract, soft tissue (e.g. mediastinum or retroperitoneum), bone marrow,joint or stomach fibrosis, in particular liver, kidney, skin, epidermis,endodermis, muscle, tendon, cartilage, heart, pancreas, lung, uterus,nervous system, testis, ovary, adrenal gland, artery, vein, colon,intestine (e.g. small intestine), biliary tract, soft tissue (e.g.mediastinum or retroperitoneum), bone marrow, joint or stomach fibrosis.

According to the present invention, the terms “cholestasis” or“cholestatic disease”, or “cholestatic disorder” and declinationsthereof denote a pathological condition defined by a decrease in bileflow due to impaired secretion by hepatocytes or to obstruction of bileflow through intra- or extrahepatic bile ducts. Therefore, the clinicaldefinition of cholestasis is any condition in which substances normallyexcreted into bile are retained.

In a particular embodiment, the fibrotic disorder is selected in thegroup consisting of a liver, gut, lung, heart, kidney, muscle, skin,soft tissue (e.g. mediastinum or retroperitoneum), bone marrow,intestinal, and joint (e.g. knee, shoulder or other joints) fibrosis.

In a preferred embodiment, the fibrotic disorder is selected in thegroup consisting of liver, lung, skin, kidney and intestinal fibrosis.

In a more preferred embodiment of the present invention, treatedfibrotic disorder is selected in the group consisting of the followingnon exhaustive list of fibrotic disorders: non-alcoholic steatohepatitis(NASH), pulmonary fibrosis. Idiopathic pulmonary fibrosis, skinfibrosis, eye fibrosis, endomyocardial fibrosis, mediastinal fibrosis,myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (acomplication of coal workers' pneumoconiosis), proliferative fibrosis,neoplastic fibrosis, lung fibrosis consecutive to chronic inflammatoryairway disease (COPD, asthma, emphysema, smoker's lung, tuberculosis),alcohol or drug-induced liver fibrosis, liver cirrhosis,infection-induced liver fibrosis, radiation or chemotherapeutic-inducedfibrosis, nephrogenic systemic fibrosis, Crohn's disease, ulcerativecolitis, keloid, old myocardial infarction, scleroderma/systemicsclerosis, arthrofibrosis, some forms of adhesive capsulitis, chronicfibrosing cholangiopathies such as Primary Sclerosing Cholangitis (PSC)and Primary Biliary Cholangitis (PBC), biliary atresia, familialintrahepatic cholestasis type 3 (PFIC3), peri-implantational fibrosisand asbestosis.

According to a particular embodiment of the invention, thecholestestatic disease is selected in the group consisting of primarybiliary cholangitis (PBC), primary sclerosing cholangitis (PSC).Intrahepatic Cholestasis of Pregnancy, Progressive Familial IntrahepaticCholestasis. Biliary atresia, Cholelithiasis, Infectious Cholangitis,Cholangitis associated with Langerhans cell histiocytosis. Alagillesyndrome, Nonsyndromic ductal paucity. Drug-induced cholestasis, andTotal parenteral nutrition-associated cholestasis. In a preferredembodiment, the cholestatic disease is PBC or PSC, in particular PBC.

The term “treatment” or “treating” refers to the curative or preventivetreatment of a cholestatic or fibrotic disorder in a subject in needthereof. The treatment involves the administration of the compound, inparticular comprised in a pharmaceutical composition, to a subjecthaving a declared disorder, i.e. to a patient, to cure, delay, reverse,or slow down the progression of the disorder, improving thereby thecondition of the subject. A treatment may also be administered to asubject that is healthy or at risk of developing a cholestatic orfibrotic disorder to prevent or delay the disorder.

Therefore, according to the invention, the treatment of a fibroticdisorder involves the administration of NTZ or TZ(G), or of apharmaceutically acceptable salt thereof, or of a pharmaceuticalcomposition containing the same, to a subject having a declared disorderto cure, delay, reverse or slow down the progression of the disorder,thus improving the condition of the patient or to a healthy subject, inparticular a subject who is at risk of developing a cholestatic orfibrotic disorder.

The subject to be treated is a mammal, preferably a human. The subjectto be treated according to the invention can be selected on the basis ofseveral criteria associated with cholestatic or fibrotic diseases suchas previous drug treatments, associated pathologies, genotype, exposureto risk factors, viral infection, as well as on the basis of thedetection of any relevant biomarker that can be evaluated by means ofimaging methods and immunological, biochemical, enzymatic, chemical, ornucleic acid detection methods.

Synthesis of NTZ or TZ can be for example carded out as described byRossignol and Caviar, 1975, or by any other way of synthesis known by aperson skilled in the art. TZG can be, for example, synthesizedaccording to way of synthesis known in the art such as in Wadouachi2011. S'agit-il de A Wadouachi, J Kovensky, Synthesis of Glycosides ofGlucuronic, Galacturonic and Mannuronic Acids: An Overview, Molecules,2011, 16(5), 3933-3968.

In a particular embodiment, the treatment of a cholestatic or fibroticdisorder may comprise the administration of a combination of both NTZand TZ(G), or of a pharmaceutically acceptable salt of NTZ and TZ(G).According to a variant of this embodiment, both NTZ and TZ(G) arecomprised together in a single composition.

In another variant of this embodiment, NTZ and TZ(G) are forsimultaneous, sequential or separate administration in therapy,therefore being possibly included in different compositions. In case ofsequential administration, NTZ may be administered prior to theadministration of TZ(G), or TZ(G) may be administered prior to NTZadministration. As such, the invention also relates to a kit-of-partscomprising (i) NTZ or a pharmaceutically acceptable salt thereof, or apharmaceutical composition comprising NTZ or a pharmaceuticallyacceptable salt thereof; and (ii) TZ(G) or a pharmaceutically acceptablesalt thereof, or a pharmaceutical composition comprising TZ(G) or apharmaceutically acceptable salt thereof, for simultaneous, sequentialor separate administration.

NTZ or TZ(G) can be formulated as pharmaceutically acceptable saltsparticularly acid or base salts compatible with pharmaceutical use.Salts of NTZ and TZ(G) include pharmaceutically acceptable acid additionsalts, pharmaceutically acceptable base addition salts, pharmaceuticallyacceptable metal salts, ammonium and alkylated ammonium salts. Thesesalts can be obtained during the final purification step of the compoundor by incorporating the salt into the previously purified compound.

In a another aspect, the present invention concerns a pharmaceuticalcomposition comprising a compound selected from NTZ or TZ(G), or apharmaceutically acceptable salt of NTZ or TZ(G), for use in a method oftreatment of a cholestatic or fibrotic disease.

The pharmaceutical composition comprising NTZ or TZ(G), in particularfor use in a method for the treatment of a cholestatic or fibroticdisorder, can also comprise one or several pharmaceutically acceptableexcipients or vehicles (e.g. saline solutions, physiological solutions,isotonic solutions, etc., compatible with pharmaceutical usage andwell-known by one of ordinary skill in the art).

These compositions can also further comprise one or several agents orvehicles chosen among dispersants, solubilisers, stabilisers,preservatives, etc. Agents or vehicles useful for these formulations(liquid and/or injectable and/or solid) are particularlymethylcellulose, hydroxymethylcellulose, carboxymethylcellulose,polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia,liposomes, etc.

These compositions can be formulated in the form of injectablesuspensions, syrups, gels, oils, ointments, pills, tablets,suppositories, powders, gel caps, capsules, aerosols, etc., eventuallyby means of galenic forms or devices assuring a prolonged and/or slowrelease. For this kind of formulations, agents such as cellulose,carbonates or starches can advantageously be used.

NTZ or TZ(G) may be administered by different routes and in differentforms. For example, the compound(s) may be administered via a systemicway, per os, parenterally, by inhalation, by nasal spray, by nasalinstillation, or by injection, such as for example intravenously, byintramuscular route, by subcutaneous route, by transdermal route, bytopical route, by intra-arterial route, etc.

Of course, the route of administration will be adapted to the form ofNTZ or TZ(G) according to procedures well known by those skilled in theart.

In a particular embodiment, the compound is formulated as a tablet. Inanother particular embodiment, the compound is administered orally.

NTZ or TZ(G), or a pharmaceutically acceptable salt thereof, isadministered in a therapeutically effective amount. Within the contextof the invention, the term “effective amount” refers to an amount of thecompound sufficient to produce the desired therapeutic effect.

The frequency and/or dose relative to the administration can be adaptedby one of ordinary skill in the art, in function of the patient, thepathology, the form of administration, etc. Typically, NTZ or TZ(G) canbe administered for the treatment of a cholestatic or fibrotic diseaseat a dose comprised between 0.01 mg/day to 4000 mg/day, such as from 50mg/day to 2000 mg/day, such as from 100 mg/day to 2000 mg/day; andparticularly from 100 mg/day to 1000 mg/day. In a particular embodiment,the NTZ, TZ(G), or a pharmaceutically acceptable salt thereof, isadministered at a dose of about 1000 mg/day (i.e at a dose of 900 to1100 mg/day), in particular at 1000 mg/day. In a particular embodiment,NTZ, TZ(G), or a pharmaceutically acceptable salt thereof, isadministered orally at a dose of about 1000 mg/day, in particular at1000 mg/day, in particular as a tablet. Administration can be performeddaily or even several times per day, if necessary. In one embodiment,the compound is administered at least once a day, such as once a day,twice a day, or three times a day. In a particular embodiment, thecompound is administered once or twice a day. In particular, oraladministration may be performed once a day, during a meal, for exampleduring breakfast, lunch or dinner, by taking a tablet comprising thecompound at a dose of about 1000 mg, in particular at a dose of 1000 mg.In another embodiment, a tablet is orally administered twice a day, suchas by administering a first tablet comprising the compound at a dose ofabout 500 mg (i.e. at a dose of 450 to 550 mg), in particular at a doseof 500 mg, during one meal, and administering a second tablet comprisingthe compound at a dose of about 500 mg, in particular at a dose of 500mg, during another meal the same day.

Suitably, the course of treatment with NTZ, TZ(G) or a pharmaceuticallyacceptable salt thereof is for at least 1 week, in particular for atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 24 weeks or more. Inparticular, the course of treatment with NTZ, TZ(G) or apharmaceutically acceptable salt thereof is for at least 1 year, 2years, 3 years, 4 years or at least 5 years.

In a particular embodiment, the invention relates to the treatment of acholestatic or fibrotic disease, in particular liver fibrosis, moreparticularly liver fibrosis consecutive to NASH, in a patient in needthereof, comprising administering to said patient a therapeuticallyeffective amount of NTZ or TZ(G) or of a pharmaceutically acceptablesalt of NTZ or TZ(G), in particular administering NTZ at a dose of 1000mg/day, in particular by administering a tablet containing 500 mg of NTZtwice a day, in particular during two different meals.

In a particular embodiment, the invention relates to the use of NTZ orTZ(G), or a pharmaceutically acceptable salt of NTZ or TZ(G) for thetreatment of a cholestatic or fibrotic disease, in combination with atleast one other therapeutically active agent with known antifibroticactivity. According to a variant of this embodiment, NTZ or TZ(G) can becombined with any antifibrotic compound such as pirfenidone or receptortyrosine kinase inhibitors (RTKIs) such as Nintedanib, sorafenib andother RTKIs, or angiotensin II (AT1) receptor blockers, or CTGFinhibitor, or any antifibrotic compound susceptible to interfere withthe TGFβ- and BMP-activated pathways including activators of the latentTGFβ complex such as MMP2, MMP9, THBS1 or cell-surface integrins. TGFβreceptors type I (TGFBRI) or type II (TGFBRII) and their ligands such asTGFβ, Activin, inhibin, Nodal, anti-Müllerian hormone, GDFs or BMPs,auxiliary co-receptors (also known as type III receptors), or componentsof the SMAD-dependent canonical pathway including regulatory orinhibitory SMAD proteins, or members of the SMAD-independent ornon-canonical pathways including various branches of MAPK signaling,TAK1, Rho-like GTPase signaling pathways, phosphatidylinositol-3kinase/AKT pathways. TGFβ-induced EMT process, or canonical andnon-canonical Hedgehog signaling pathways including Hh ligands or targetgenes, or any members of the WNT, or Notch pathways which aresusceptible to influence TGFβ signaling.

Thus, the invention also relates to a pharmaceutical compositioncomprising a compound selected from NTZ or TZ(G), or a pharmaceuticallyacceptable salt of NTZ or of TZ(G), in combination with at least onetherapeutically active agent with known antifibrotic activity selectedfrom pirfenidone or receptor tyrosine kinase inhibitors (RTKIs) such asNintedanib, sorafenib and other RTKIs, or angiotensin II (AT1) receptorblockers, or CTGF inhibitor, or antifibrotic compound susceptible tointerfere with the TGFβ- and BMP-activated pathways including activatorsof the latent TGFβ complex such as MMP2, MMP9, THBS1 or cell-surfaceintegrins, TGFβ receptors type I (TGFBRI) or type II (TGFBRII) and theirligands such as TGFβ, Activin, inhibin, Nodal, anti-Müllerian hormone,GDFs or BMPs, auxiliary co-receptors (also known as type III receptors),or components of the SMAD-dependent canonical pathway includingregulatory or inhibitory SMAD proteins, or members of theSMAD-independent or non-canonical pathways including various branches ofMAPK signaling, TAK1, Rho-like GTPase signaling pathways,phosphatidylinositol-3 kinase/AKT pathways. TGFβ-induced EMT process, orcanoninal and non-canonical Hedgehog signaling pathways including Hhligands or target genes, or any members of the WNT, or Notch pathwayswhich are susceptible to influence TGFβ signaling, for use in a methodfor treating a fibrotic disorder.

In another particular embodiment, other classes of molecules that couldalso be combined with NTZ or TZ(G) include JAK/STAT inhibitors, or otheranti-inflammatory and/or immunosuppressant agents. A non exhaustive listof these agents includes but is not limited to glucocorticoids, NSAIDS,cyclophosphamide, nitrosoureas, folic acid analogs, purine analogs,pyrimidine analogs, methotrexate, azathioprine, mercaptopurine,ciclosporin, myriocin, tacrolimus, sirolimus, mycophenolic acidderivatives, fingolimod and other sphingosine-1-phosphate receptormodulators, monoclonal and/or polyclonal antibodies against such targetsas proinflammatory cytokines and proinflammatory cytokine receptors,T-cell receptor, integrins. Other classes of molecules that could alsobe combined with NTZ or TZ(G) include molecules that could potentiallyenhance the exposure or the effect of NTZ or TZ(G).

In another particular embodiment, the invention relates to a combinationof NTZ, TZ(G) or a pharmaceutically acceptable salt thereof with atleast one other therapeutically active agent with known antifibroticactivity, or with JAK/STAT inhibitors, or other anti-inflammatory and/orimmunosuppressant agents. The combination may be in the form of a singlepharmaceutical composition comprising the different active ingredients,including NTZ, TZ(G), or a pharmaceutically acceptable salt thereof. Ina variant, the combination is a kit of parts comprising NTZ, TZ(G), or apharmaceutically acceptable salt thereof, and another active ingredientsuch as another anti-fibrotic agent, a JAK/STAT inhibitor, or anotheranti-inflammatory or immunosuppressant agent. Said kit of parts may befor simultaneous, separate or sequential administration for thetreatment of a cholestatic or fibrotic disorder

In another embodiment, compound NTZ or TZ(G), or a combination of NTZand TZ(G), is administered as the sole active ingredient. Accordingly,the invention also relates to a pharmaceutical composition comprising acompound selected from NTZ or TZ(G), or a pharmaceutically acceptablesalt of NTZ or of TZ(G), or a mixture thereof, for use in a method fortreating a cholestatic or fibrotic disorder, wherein said compound(s) is(are) the only active ingredient(s) in the composition

In a further embodiment, the present invention provides methods oftreating cholestatic or fibrotic diseases comprising the administrationof NTZ or TZ(G), or a pharmaceutically acceptable salt of NTZ or TZ(G),in particular in the form of a pharmaceutical composition containing NTZor TZ.

In another aspect, the invention relates to a kit-of-parts comprising:

-   -   Nitazoxanide or a pharmaceutically acceptable salt of        Nitazoxanide; and    -   Tizoxanide or a pharmaceutically acceptable salt of Tizoxanide.

The compounds of the kit of parts of the invention are administeredsimultaneously, separately or sequentially for the treatment of afibrotic disorder.

In another embodiment, the invention provides a method of treating acholestactic and/or fibrotic diseases comprising administering twicedaily to a patient in need thereof having a cholestatic or fibroticdisease (in particular to a NASH patient or to a patient having a liverfibrosis) a tablet containing 500 mg of NTZ, in particular during a meal(such as during breakfast, lunch or dinner).

The invention is further described with reference to the following,non-limiting, examples.

EXAMPLES

Materials & Methods

Compounds were dissolved in dimethyl sulfoxide (DMSO, Fluka cat#41640).Nitazoxanide (INTERCHIM cat#RQ550U) and Tizoxanide (INTERCHIM cat#RP253)were obtained commercially.

hHSC Culture

The human primary hepatic stellate cells (hHSC) (Innoprot) were culturedin STeCM medium (ScienCell cat#5301) that was supplemented with 2% fetalbovine serum (FBS, ScienCell cat#0010), 1% penicillin/streptomycin(ScienCell cat#0503) and stellate cell growth supplement (SteCGS;ScienCell cat#5352). Cell culture flasks were coated with Poly-L Lysine(Sigma cat# P4707) for a better adherence.

Activation of hHSC with TGF-β1

The human primary hepatic stellate cells (hHSC) (Innoprot) were culturedunder standard conditions, as described above. The cells weresubsequently plated at a density of 7×10⁴ cells/well into 24-well platesfor gene expression studies, and at a density of 2×10⁴ cells/well into96-well plates for the measure of α-SMA by ELISA. The next day, cellculture medium was removed, and cells were washed with PBS (Invitrogencat#14190). hHSC were deprived for 24 hours in serum-free andSteCGS-free medium. For the treatments with NTZ or TZ, theserum-deprived hHSC were preincubated for 1 hour with the compoundsfollowed by the addition of the profibrogenic stimuli TGFβ1 (PeproTechcat#100-21, 1 ng/mL) in serum-free and SteCGS-free medium for anadditional 24 or 48 hour period (the timepoint is indicated in thefigure legends). At the end of treatment, cells were washed with PBS(Invitrogen, cat#14190) before the addition of 50 μl of lysis buffer(CelLytic™ MT reagent; Sigma #C3228). Plates were then incubated for 30min on ice using a plate shaker, before storage at −20° C.

Activation of Rat HSC with TGFβ1:

The rat primary hepatic stellate cells (rHSC) (Innoprot) were culturedin STeCM medium (ScienCell cat#5301) that was supplemented with 2% fetalbovine serum (FBS. ScienCell cat#0010), 1% penicillin/streptomycin(ScienCell cat#0503) and stellate cell growth supplement (SteCGS;ScienCell cat#5352). For the activation experiments with TGFβ1, the rHSCwere plated at a density of 10×10³ cells per well in 96-well plates. Thenext day, cell culture medium was removed, and cells were washed withPBS (Invitrogen cat#14190). rHSC were deprived for 24 hours inserum-free and SteCGS-free medium. For the treatments with NTZ or TZ,the serum-deprived rHSC were preincubated for 1 hour with the compoundsfollowed by addition of the profibrogenic stimuli TGFβ1 (PeproTechcat#100-21, 3 ng/mL) in serum-free and SteCGS-free medium for anadditional 48 hour period. At the end of treatment, cells were washedwith PBS (Invitrogen, cat#14190) before the addition of 50 μl of lysisbuffer (CelLytic™ MT reagent; Sigma #C3228). Plates were then incubatedfor 30 min on ice using a plate shaker, before storage at −20° C.

Activation of NHLF with TGFβ1

The Normal Human Lung Fibroblasts (NHLF) (Lonza) were cultured inFibroblast Basal Medium (FBM) (Lonza cat#CC-3131) that was supplementedwith FGM-2 SingleQuots™ Kit (Lonza cat#CC-3132). The complete mediumcontains 2% fetal bovine serum. For the activation experiments withTGFβ1, the NHLF were plated at a density of 5×10³ cells per well in96-well plates. The next day, cell-culture medium was removed, and cellswere washed with PBS (Invitrogen cat#14190). NHLF were deprived for 24hours in serum-free, insulin-free and rhFGF-B-free medium. For thetreatments with NTZ or TZ, the serum-deprived NHLF were preincubated for1 hour with the compounds followed by addition of the profibrogenicstimuli TGFβ1 (PeproTech cat#100-21, 1 ng/mL) in serum-free,insulin-free and rhFGF-B-free medium for an additional 48 hour period.At the end of treatment, cells were washed with PBS (Invitrogen,cat#14190) before the addition of 50 μl of lysis buffer (CelLytic™ MTreagent; Sigma #C3228). Plates were then incubated for 30 min on iceusing a plate shaker, before storage at −20° C.

Activation of NHCF-V with TGFβ1:

The Normal Human Cardiac Fibroblasts (ventricle) (NHCF-V) (Lonza) wereisolated from normal adult heart tissue. Cells were cultured inFibroblast Basal Medium (FBM) (Lonza cat#CC-3131) that was supplementedwith FGM™-3 BulletKit™ kit (Lonza cat#CC-4525). The complete mediumcontains 10% fetal bovine serum. For the activation experiments withTGFβ1, the NHCF-V were plated at a density of 6×10³ cells per well in96-well plates. The next day, cell-culture medium was removed, and cellswere washed with PBS (Invitrogen cat#14190). NHCF were deprived for 24hours in serum-free, insulin-free and rhFGF-B-free medium. For thetreatments with NTZ or TZ, the serum-deprived NHCF were preincubated for1 hour with the compounds followed by addition of the profibrogenicstimulus TGFβ1 (PeproTech cat#100-21, 3 ng/mL) in serum-free,insulin-free and rhFGF-B-free medium for an additional 48 hour period.At the end of treatment, cells were washed with PBS (Invitrogen,cat#14190) before the addition of 50 μl of lysis buffer (CelLytic™ MTreagent; Sigma #C3228). Plates were then incubated for 30 min on iceusing a plate shaker, before storage at −20° C.

Activation of InMyoFib with TGFβ1:

The Human Intestinal Myofibroblasts (InMyoFib) (Lonza) were cultured inSmooth Muscle Cell Basal Medium (SmBM-2TM) (Lonza cat#CC-3181) that wassupplemented with SmGMTM-2 BulletKit™ (Lonza cat#CC-4149). The completemedium contains 5% fetal bovine serum. For the activation experimentswith TGFβ1, the inMyoFib were plated at a density of 10×10³ cells perwell in 96-well plates. The next day, cell-culture medium was removed,and cells were washed with PBS (Invitrogen cat#14190). InMyoFib weredeprived for 24 hours in serum-free, insulin-free and rhFGF-B-freemedium. For the treatments with NTZ or TZ, the serum-deprived InMyoFibwere preincubated for 1 hour with the compounds followed by addition ofthe profibrogenic stimuli TGFβ1 (PeproTech cat#100-21, 3 ng/mL) inserum-free, insulin-free and rhFGF-B-free medium for an additional 48hour period. At the end of treatment, cells were washed with PBS(Invitrogen, cat#14190) before the addition of 50 μl of lysis buffer(CelLytic™ MT reagent; Sigma #C3228). Plates were then incubated for 30min on ice using a plate shaker, before storage at −20° C.

α-SMA ELISA

The level of α-SMA was measured using a Sandwich ELISA. Briefly, thewells of an ELISA plate were first coated with the capture antibody(mouse monoclonal anti-ACTA2, Abnova) at 4° C. overnight. After 3 washesin PBS+0.2% Tween 20, a blocking solution consisting of PBS+0.2% BSA wasadded for one hour followed by another washing cycle. The cell lysateswere transferred into the wells for binding to the capture antibody fora period of 2 h at room temperature. After the washing procedure, thedetection antibody (blotinylated mouse monoclonal anti-ACTA2, Abnova)was added for 2 hours at room temperature followed by 3 washes. For thedetection, an HRP-conjugated Streptavidin (R&D Systems cat#DY998) wasfirst applied for 30 min at room temperature. After washing, the HRPsubstrate TMB (BD, #555214) was added and incubated for 7 min at roomtemperature in the dark. Upon oxidation, TMB forms a water-soluble bluereaction product that becomes yellow with addition of sulfuric acid(solution stop), enabling accurate measurement of the intensity at 450nm using a spectrophotometer. The developed color is directlyproportional to the amount of α-SMA present in the lysate.

Gene Expression

Total RNA was isolated using Nucleospin® 96 RNA (Macherey Nagel)following manufacturer's instructions. Total RNA (500 ng for in vitrosamples) were reverse transcribed into cDNA using M-MLV RT (MoloneyMurine Leukemia Virus Reverse Transcriptase) (Invitrogen cat#28025) in1×RT buffer (Invitrogen), 1 mM DTT (Invitrogen), 0.18 mM dNTPs(Promega), 200 ng pdN6 (Amersham) and 30 U of RNase inhibitor (Promega).

Quantitative PCR was then carried out using the MyiQ Single-ColorReal-Time PCR Detection System (Biorad). Briefly, the PCR reactions wereperformed in 96-WP format in 25 ul of total volume containing 1 μL ofreverse transcription reaction, 0.5 μL of reverse and forward primers(10 pmol each), and 12.5 μl of 2×iQ SYBR Green Supermix (BioRad,1725006CUST). The sequences of primers are depicted in the table 1

TABLE 1 Human Primers Primer name Sequence (5′→3′) 36B4 forwardCATGCTCAACATCTCCCCCTTCTCC (SEQ ID NO: 1) 36B4 reverseGGGAAGGTGTAATCCGTCTCCACAG (SEQ ID NO: 2) COL1A1 forwardAGGCGAACAAGGTGACAGAG (SEQ ID NO: 3) COL1A1 reverse GCCAGGAGAACCAGCAGAG(SEQ ID NO: 4)

Expression levels were normalized using the expression of 3684 gene as areference in human samples.

For each gene, the standard curves were drawn by selecting the bestpoints (at least three points) in order to have PCR reaction efficiencyclose to 100% and a correlation coefficient close to 1. Expressionlevels were determined using the standard curve equation for both thehousekeeping gene and the target gene (taking into account the specificPCR efficiency of each target gene).

Evaluation of NTZ in a Chronic CDAAc Diet-Induced Liver Fibrosis Model

The antifibrotic effect of NTZ was assessed in a murine model of CDAAcdiet-induced experimental liver fibrosis. 6 week-old C57Bl/6 mice werefed for 12 weeks a control (CSAA) diet, CDAAc diet, or CDAAc dietsupplemented with NTZ 10 mg/kg/day for 12 weeks.

The body weight and the food intake were monitored twice per week. Onthe last day of treatment, mice were sacrificed after a 6 h fastingperiod. The liver was rapidly excised for biochemical and histologicalstudies.

All animal procedures were performed according to standard protocols andin accordance with the standard recommendations for the proper care anduse of laboratory animals.

Evaluation of NTZ in CCl4-Induced Liver Damage Model

The antifibrotic effect of NTZ was assessed in a rat model of CCl4induced liver injury.

OFA S:Dawley rats (initial body weight 250-275 g) were randomizedaccording to their body weight into 4 groups and treated for 3 weeks.The rats were intraperitoneally injected with olive oil (ctrl group) orwith CCl4 emulsified in olive oil (CCl4:olive oil 1:2 v/v, final CCl4concentration: 2 ml/kg) twice weekly. Concomitantly, the olive oilinjected group was placed on control diet while the CCl4 injected groupswere placed on control diet or diet supplemented with NTZ. 2 regimencontaining NTZ were prepared corresponding respectively to an exposureof 10, or 30, mg/kg/day. The last day of treatment, the rats weresacrificed after a 6 h fasting period. Blood samples were collected andthe serum was isolated for biochemical analyses.

Evaluation of NTZ in the DDC Model of Cholestasis:

C57Bl/6 mice will be fed for 8 weeks a 0.1% DDC-supplemented diet, or0.1% DDC-supplemented diet containing NTZ 100 mg/kg/day, or a standardmouse diet (Ssniff). The last day of treatment, the mice will besacrificed after a 6 h fasting period. Blood samples will be taken forbiochemical analyses and the liver will be rapidly excised forbiochemical and histological studies.

Evaluation of NTZ in a Chronic CCl4-Induced Liver Fibrosis Model

9 week-old C57Bl/6 mice will be placed on control diet or dietsupplemented with NTZ for 6 weeks. 2 regimen containing NTZ will beprepared corresponding respectively to an exposure of NTZ 30, or 100mg/kg/day. Concomitantly, and for the total duration of 6 weeks, themice will be treated 3 times a week with CCl4 dissolved in olive oil orvehicle by oral gavage. The amount of C014 will be progressivelyincreased from 0.875 ml/kg to 2.5 ml/kg. The last day of treatment, themice will be sacrificed after a 6 h fasting period. Blood samples willcollected for biochemical analyses of serum. The liver will be rapidlyexcised for biochemical, histological and expression studies.

Histology

Tissue Embedding and Sectioning:

The liver slices were first fixed for 12 hours in a solution of 4%formalin. The, liver pieces were then washed 30 minutes in PBS, anddehydrated in ethanol solutions (successive baths at 70, 80, 95 and 100%ethanol). The liver pieces were incubated in three different baths ofXylene (Sigma-Aldrich cat#534056), followed by two baths in liquidparaffin (56° C.). Liver pieces were then put into racks that weregently filled with Histowax® to completely cover the tissue.

The paraffin blocks containing the tissue pieces were removed from theracks and stored at room temperature. The liver blocks were cut into 3μm slices.

Picrosirius Red Staining

Liver sections were deparaffinized, rehydrated and incubated for 15minutes in a solution of Fast Green FCF 0.1% (Sigma-Aldrich, cat#F7258)before rinsing in a bath of 0.5% acetic acid (Panreac, cat#131008.1611).The liver sections were rinsed in water and incubated for 30 minutes ina solution of 0.1% sirius red (Direct Red 80, Fluka cat#43665) insaturated aqueous picric acid (Sigma-Aldrich cat#P6744). The liversections were finally dehydrated, and mounted using the CV Mount medium(Leica, cat #14046430011).

Histological Examinations

The identity of the liver specimen was blinded from the examiner.Virtual slides were generated using the Pannoramic 250 scanner from 3DHistech. Using Quant Center software (3D Histech, including PatternQuant and Histo Quant modules), collagen-stained areas were quantified.Briefly, Pattern Quant was used to detect relevant tissue structure andto measure the surface. Then, Histo Quant was used to detect the stainedcollagen content and for the measurement of total area and percentages,based on a color threshold method. The fibrosis area was expressed asthe percentage of collagen surface over the whole tissue.

Measurement of Hepatic Collagen Content

The hepatic collagen content was determined using the appropriateQuickZyme kit (Total collagen assay, cat#QZB-totcol2). The assay isbased on the detection of hydroxyproline, which is a non-proteinogenicamino acid mainly found in the triple helix of collagen. Thus,hydroxyproline in tissue hydrolysates can be used as a direct measure ofthe amount of collagen present in the tissue (without discriminationbetween procollagen, mature collagen and collagen degradation products).

Complete hydrolysis of tissue samples in 6M HCl at 95° C. is requiredbefore dosing the hydroxyproline. The assay results in the generation ofa chromogen with a maximum absorbance at 570 nm. Results are expressedas mg of collagen/g of liver.

Evaluation of NTZ in the BDL Model

Surgical bile duct ligation will be performed on rats in order to inducean extrahepatic cholestasis and subsequently liver fibrosis. After a 2week recovery period, animals will be treated with NTZ at 30 or 100mg/kg/day for one or two weeks. The last day of treatment, the mice willbe sacrificed after a 6 h fasting period. Blood samples will becollected for biochemical analyses of serum. The liver will be rapidlyexcised for biochemical, histological & expression studies.

Measurement of Plasmatic Concentration of Total Bile Acids

The plasmatic concentration of Total Bile Acids (TBA) was determinedusing the appropriate Randox kit for the Daytona automated analyzer(Randox, cat#BI 3863). In the presence of Thio-NAD, the enzyme 3-αhydroxysteroid dehydrogenase (3-α HSD) converts bile acids to 3-ketosteroids and Thio-NADH. The reaction is reversible and 3-α HSD canconvert 3-ketosteroids and Thio-NADFH-to bile acids and Thio-NAD. In thepresence of excess NADH, the enzyme cycling occurs efficiently and therate of formation of Thio-NADH is determined by measuring specificchange of absorbance at 405 nm. Results are expressed in pmol/L.

Results and Conclusions:

The abnormal persistence of differentiated myofibroblasts is acharacteristic of many fibrotic diseases. Following liver injury,quiescent HSC undergo a process of activation that is characterized by adifferentiation into (α-SMA)-positive myofibroblasts. In an attempt tofind new antifibrotic molecules, a library of FDA-approved drugs wasphenotypically screened in a model of human HSC activated with theprofibrogenic cytokine TGFβ1. The levels of α-SMA, a hallmark offibrotic lesions, were used to evaluate the potency of the drugs tointerfere with the fibrotic process. The screening campaign led to theidentification of nitazoxanide (NTZ), which dose-dependently reduced thelevel of α-SMA in TGFβ-induced HSCs. Overall, NTZ exhibited an IC₅₀comprised between 0.1 and 3 μM (FIG. 1A). Since it is known that NTZ israpidly hydrolyzed into an active metabolite tizoxanide (TZ)(Broekhuysen, Stockis et al., 2000), this metabolite was also evaluatedfor its antifibrotic activity in HSC. TZ showed a profile similar to theparent drug with an IC₅₀ comprised between 0.1 and 3 μM (FIG. 1B). Othermarkers of TGFβ stimulation were reduced by both compounds including theextracellular matrix collagen 1A1 (COL1A1) (FIG. 2). Toxicity assaysconfirmed that the reduced levels of α-SMA were not due to toxicity orapoptosis of HSC (data not shown).

NTZ and TZ also reduced α-SMA levels in TGFβ-activated HSC derived fromrat (FIG. 3). In addition, the antifibrotic potential of NTZ and TZ wasextended to fibroblasts derived from other tissues, including normalhuman lung fibroblasts (NHLF) (FIG. 4), normal human cardiac fibroblasts(FIG. 5) and human Intestinal myofibroblasts (InMyoFib) (FIG. 6). In allthese models of fibrosis, NTZ and TZ showed significant antifibroticeffects at a concentration of 1 μM.

In vivo efficacy of NTZ was assessed in a CDAA cholesterol diet-inducedexperimental liver fibrosis model. The chronic oral administration ofNitazoxanide 10 mg/kg/day demonstrated antifibrotic properties reflectedby significantly lower hepatic collagen content (FIG. 7) and reducedhepatic fibrosis area by histological examination (FIG. 8).

In the in vivo model of CCl₄-induced liver injury, NTZ prevented thepathological increase of circulating TBA concentration (FIG. 9), whichis a marker associated with cholestasis (Chang 2013)

In conclusion, the applicant has discovered unexpected antifibrotic andanticholestatic activities for the antiparasitic agent NTZ. Theseresults demonstrate that NTZ and/or its active metabolite TZ can providetherapeutic benefits in cholestatic diseases and multiple types offibrotic diseases.

REFERENCES

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The invention claimed is:
 1. A method for treating heart fibrosiscomprising administering to a subject in need thereof a therapeuticeffective amount of a compound selected in the group consisting ofNitazoxanide (NTZ), Tizoxanide (TZ), and a pharmaceutically acceptablesalt of NTZ or TZ.
 2. The method according to claim 1, wherein thecompound is administered orally.
 3. The method according to claim 1,wherein the compound is administered in an amount comprised between 100mg/day and 2000 mg/day.
 4. The method according to claim 1, wherein thecompound is administered in the form of a pharmaceutical compositionproviding prolonged or slow release of the compound.
 5. The methodaccording to claim 1, wherein heart fibrosis is endomyocardial fibrosisor fibrosis consecutive to myocardial infarction.
 6. A method fortreating heart fibrosis comprising administering to a subject in needthereof a therapeutic effective amount of nitazoxanide.
 7. The methodaccording to claim 6, wherein nitazoxanide is administered orally. 8.The method according to claim 6, wherein nitazoxanide is administered inan amount comprised 100 mg/day and 2000 mg/day.
 9. The method accordingto claim 6, wherein nitazoxanide is administered in the form of apharmaceutical composition providing prolonged or slow release ofnitazoxanide.
 10. The method according to claim 6, wherein heartfibrosis is endomyocardial fibrosis or fibrosis consecutive tomyocardial infarction.